Gelofusine As Alternative to Dextran40 for Washing Cryopreserved Hematopoietic Stem Cell Products Prior to Infusion: Validation and Application to Clinical Practice

Background

Cryopreservation represents an important step in autologous hematopoietic stem cell transplantation, and when allogeneic peripheral blood stem cells (PBSC) or umbilical cord blood units (CBUs) and sometimes bone marrow (BM) cannot be immediately infused after harvest and must be stored.

Hematopoietic stem cells (HSCs) are cryopreserved in dimethyl sulfoxide (DMSO). Although DMSO preserves cell viability, it can be toxic to cells after thawing and has a dose-dependent toxicity during infusion. Cryopreserved HSC thawing and washing procedures represent a key factor for the efficiency of the transplant. A validated solution to wash cryopreserved cells was proposed by Rubinstein et al., but dextran40, the main component, became unavailable in Italy. There are no published alternative solutions available.

Materials and methods

We report our cell processing laboratory experience with gelofusine, a 4% modified fluid gelatin colloidal volume replacement solution, as alternative solution to wash cell products at the Santobono Pausilipon Hospital, Naples IT.

We report: 1) validation of gelofusine solution in 10 CBUs; 2) outcomes (TNC and CD34+ recovery and viability after thawing, infusion reactions, time to engraftment) for the first 62 products using the gelofusine solution in pediatric and adult patients; 3) comparisons of TNC and CD34⁺ recovery and viability in 5 paired autologous PBSC products cryopreserved with the 2 washing solutions (dextran40 vs. gelofusine) in adult patients planned to undergo sequential transplants; and 4) comparisons of ANC and platelet engraftment for the 2 different solutions in cell products infused in 1 of 2 populations of 29 patients with similar clinical features.

The cryoprotectant solutions consisted of 10% DMSO for all products. Stem cells were frozen using a rate-controlled freezer and stored in liquid nitrogen. The products were thawed rapidly in a 37°C water bath, and washed with the method reported by Rubinstein et al. or with automated method using the Biosafe Sepax system. The standard wash solution, proposed by Rubinstein et al., was prepared from 10% dextran40 in saline (5 parts) diluted with 25% HSA (1 part) giving a final concentration of 5% dextran40 and 2.5% HSA. The same wash solution replacing gelofusine for dextran40 was prepared. TNC count was performed using the Sysmex XN-1000. To assess CD34⁺ and TNC viability and CD34⁺ count was used FACSCanto II flow cytometer using Enumeration kit Stem Cell-BD.

Cell recovery (%) was based on cryopreservation values. Viability (%) was determined by analyzing both 7AAD negative (viable) and positive (dead) populations.

Results were express as mean ± standard deviation and compared using a paired t-test. A p-value of less than 0.05 was considered significant.

Results

1) CD34⁺ viability and recovery and TNC recovery were 96±2, 87±12, 75±12, respectively for the 10 CBUs washed with gelofusine, higher than our reference values (TNC recovery ≥ 40%, CD34⁺ recovery ≥50%, CD34⁺ viability ≥70%).

2) CD34+ and TNC recovery were 89±8, 96±2 and viability were 85±8, 92±3, respectively for the 62 products washed with gelofusine, higher than our reference values. One patient experienced infusion reaction; all patients engrafted.

3) The 5 paired autologous PBSC products showed comparable results between dextran40 and gelofusine regarding TNC and CD34+ recovery (98±1 vs. 98±2, p=0.76; 78±3 vs. 85±9, p=0.14), while a significant difference was observed regarding TNC viability (68±9 vs. 89±0.4, p=0.005) and CD34+ viability (89±2 vs. 97±1, p=0.001).

4) The comparison between the 2 groups of 29 patients showed time to ANC recovery at day 12.9±5.0 and at day 11.7±3.0 (p=0.10) and platelet engraftment at day 13.2±2.4 and 12±3.2 (p=0.19) for patients who received dextran40 and gelofusine washed products, respectively

Conclusion

To our knowledge, this is the first study proposing an alternative solution for washing cryopreserved HSCs. The findings apply to different HSCs sources such as PBSC, BM and CBU, different in cellular composition, and to adult and pediatric patients. The data suggest that gelofusine is an alternative to dextran40 for washing solution for cryopreserved HSCs.

Picardi:NOVARTIS: Other: Advisory board; MEDAC: Speakers Bureau; AMGEN: Speakers Bureau; MSD: Other: Advisory board; GILEAD: Speakers Bureau. Wierda:Novartis: Research Funding; National Comprehensive Care Center (NCCN): Other: Financial relationship (Chair, CLL); Nurix Therapeutics: Research Funding; BMS: Research Funding; Oncternal Therapeutics: Research Funding; Kite: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; F. Hoffmann-La Roche Ltd.: Research Funding; AstraZeneca: Research Funding; Acerta Pharma: Research Funding; Janssen: Research Funding; Oncternal Therapeutics: Research Funding; Accutar Biotechnology: Research Funding; Genentech, Inc.: Research Funding; Cyclacel Pharmaceuticals Inc: Research Funding; GSK: Research Funding; Gilead Sciences: Research Funding; Numab Therapeutics: Research Funding; Juno Therapeutics: Research Funding; Loxo Oncology: Research Funding; Eli Lilly: Research Funding; AbbVie: Research Funding. Tambaro:Jazz Pharma: Membership on an entity's Board of Directors or advisory committees, Other: travel expense; Amgen: Other: travel expense, Speakers Bureau; Novartis: Other: travel support; Neovii: Other: travel support ; Gilead: Membership on an entity's Board of Directors or advisory committees; Medac: Other: travel support , Speakers Bureau.